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1.
Cell ; 152(3): 532-42, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23374348

RESUMO

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.


Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Transdução de Sinais , Termodinâmica
2.
Nature ; 535(7610): 182-6, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27362234

RESUMO

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR­G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.


Assuntos
Sítio Alostérico , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/farmacologia , Guanina/metabolismo , Guanina/farmacologia , Humanos , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores Adrenérgicos beta 2/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia
3.
Biophys J ; 115(2): 300-312, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30021106

RESUMO

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (ß2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.


Assuntos
Multimerização Proteica , Proteolipídeos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Estrutura Quaternária de Proteína , Transdução de Sinais , Solubilidade
4.
Nature ; 492(7429): 387-92, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23222541

RESUMO

Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.


Assuntos
Receptor PAR-1/química , Motivos de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Ativação Enzimática/genética , Humanos , Hidrólise , Lactonas/química , Lactonas/farmacologia , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Infarto do Miocárdio/prevenção & controle , Conformação Proteica , Piridinas/química , Piridinas/farmacologia , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores de Trombina
5.
Nat Methods ; 11(9): 931-4, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25086504

RESUMO

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the ß2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.


Assuntos
Algoritmos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Proteolipídeos/química , Espectrometria de Fluorescência/métodos , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/química
6.
Nature ; 469(7329): 175-80, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-21228869

RESUMO

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human ß(2) adrenergic receptor (ß(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive ß(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Nanoestruturas/química , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/imunologia , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animais , Sítios de Ligação , Camelídeos Americanos , Cristalografia por Raios X , Agonismo Inverso de Drogas , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Fragmentos de Imunoglobulinas/farmacologia , Ligantes , Modelos Moleculares , Movimento/efeitos dos fármacos , Opsinas/agonistas , Opsinas/química , Opsinas/metabolismo , Propanolaminas/química , Propanolaminas/metabolismo , Propanolaminas/farmacologia , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/metabolismo
7.
Nature ; 463(7277): 108-12, 2010 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-20054398

RESUMO

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.


Assuntos
Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Cristalografia por Raios X , Agonismo Inverso de Drogas , Etanolaminas/farmacologia , Fumarato de Formoterol , Humanos , Ligantes , Lisina/análogos & derivados , Lisina/metabolismo , Metilação , Modelos Moleculares , Proteínas Mutantes , Ressonância Magnética Nuclear Biomolecular , Propanolaminas/metabolismo , Propanolaminas/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Eletricidade Estática , Especificidade por Substrato
8.
EMBO J ; 28(21): 3315-28, 2009 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-19763081

RESUMO

The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.


Assuntos
Bicamadas Lipídicas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Cisteína/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Lipossomos/metabolismo , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Multimerização Proteica , Receptores Adrenérgicos beta 2/análise , Receptores Adrenérgicos beta 2/genética
9.
PLoS One ; 18(10): e0287863, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37878624

RESUMO

Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits ß-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (ß2AR). Co-treatment with CXCL12 and the ß2AR agonist epinephrine synergistically increases ß-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Mieloma Múltiplo , Animais , Camundongos , Mobilização de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/tratamento farmacológico , Propranolol/uso terapêutico , Cálcio/metabolismo , Compostos Heterocíclicos/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Receptores CXCR4/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , beta-Arrestinas/metabolismo , Benzilaminas/metabolismo
10.
Proc Natl Acad Sci U S A ; 106(16): 6843-8, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19342484

RESUMO

Aminergic G protein-coupled receptors (GPCRs) have been a major focus of pharmaceutical research for many years. Due partly to the lack of reliable receptor structures, drug discovery efforts have been largely ligand-based. The recently determined X-ray structure of the beta(2)-adrenergic receptor offers an opportunity to investigate the advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR targets. Approximately 1 million commercially available, "lead-like" molecules were docked against the beta(2)-adrenergic receptor structure. On testing of 25 high-ranking molecules, 6 were active with binding affinities <4 microM, with the best molecule binding with a K(i) of 9 nM (95% confidence interval 7-10 nM). Five of these molecules were inverse agonists. The high hit rate, the high affinity of the most potent molecule, the discovery of unprecedented chemotypes among the new inhibitors, and the apparent bias toward inverse agonists among the docking hits, have implications for structure-based approaches against GPCRs that recognize small organic molecules.


Assuntos
Descoberta de Drogas , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2 , Sítios de Ligação , Ligação Competitiva , Cátions , Bases de Dados de Proteínas , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Cinética , Ligantes , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/farmacologia
11.
Methods Mol Biol ; 552: 253-68, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19513655

RESUMO

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Förster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Humanos , Receptores Adrenérgicos beta 2/metabolismo , Espectrometria de Fluorescência
12.
Elife ; 62017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28653617

RESUMO

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.


Assuntos
Antineoplásicos/administração & dosagem , Cromatina/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Leucemia Aguda Bifenotípica/tratamento farmacológico , Proteínas do Tecido Nervoso/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Resultado do Tratamento
13.
Elife ; 42015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26327698

RESUMO

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Inhibition of bromodomain and extra terminal (BET) recruitment to chromatin as an effective treatment for mixed-lineage leukemia (MLL)-fusion leukemia' by Dawson and colleagues, published in Nature in 2011 (Dawson et al., 2011). The experiments to be replicated are those reported in Figures 2A, 3D, 4B, 4D and Supplementary Figures 11A-B and 16A. In this study, BET proteins were demonstrated as potential therapeutic targets for modulating aberrant gene expression programs associated with MLL-fusion leukemia. In Figure 2A, the BET bromodomain inhibitor I-BET151 was reported to suppress growth of cells harboring MLL-fusions compared to those with alternate oncogenic drivers. In Figure 3D, treatment of MLL-fusion leukemia cells with I-BET151 resulted in transcriptional suppression of the anti-apoptotic gene BCL2. Figures 4B and 4D tested the therapeutic efficacy of I-BET151 in vivo using mice injected with human MLL-fusion leukemia cells and evaluated disease progression following I-BET151 treatment. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.


Assuntos
Cromatina/metabolismo , Leucemia Aguda Bifenotípica/terapia , Animais , Feminino , Humanos , Masculino , Camundongos SCID , Reprodutibilidade dos Testes , Resultado do Tratamento
14.
PeerJ ; 3: e1232, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26401448

RESUMO

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

15.
Protein Eng Des Sel ; 27(4): 97-109, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24586053

RESUMO

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Sistema Livre de Células , Fragmentos Fab das Imunoglobulinas , Biblioteca de Peptídeos , Anticorpos/genética , Anticorpos Monoclonais Humanizados/genética , Antígeno Carcinoembrionário/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Trastuzumab , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
J Biol Chem ; 282(18): 13895-905, 2007 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-17347144

RESUMO

The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.


Assuntos
Antagonistas Adrenérgicos beta/química , Transferência Ressonante de Energia de Fluorescência , Propanolaminas/química , Receptores Adrenérgicos beta 2/química , Antagonistas Adrenérgicos beta/metabolismo , Motivos de Aminoácidos , Animais , Arrestina/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Ligantes , Propanolaminas/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
18.
Nat Methods ; 4(11): 927-9, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17952087

RESUMO

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.


Assuntos
Anticorpos Monoclonais/imunologia , Receptores Adrenérgicos beta 2/imunologia , Receptores Acoplados a Proteínas G/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Reações Antígeno-Anticorpo/imunologia , Western Blotting , Cristalização/métodos , Cristalografia , Epitopos/química , Epitopos/imunologia , Corantes Fluorescentes/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Rodaminas/química , Vacinação
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